There is no robust cell culture system in which the hepatitis C virus (HCV) can replicate. As such, surrogate models of HCV replication are frequently used for the evaluation of antiviral compounds. The GB virus B (GBV-B) is the closest related virus phylogenetically with HCV and is an attractive surrogate model for HCV drug development efforts. Unfortunately, GBV-B can so far only be grown in primary hepatocytes taken from small non-human primates. This makes the current GBV-B antiviral assay difficult to perform, expensive, and not amenable to high-throughput (HTP) screening. This application aims to develop a robust cell culture model of GBV-B replication utilizing immortalized cell lines. The specific aims of this project are to immortalize the primary hepatocytes of GBV-B-susceptible animals, identify if these and other established cell lines can support GBV-B replication in vitro, and to standardize and validate the model system produced for use in the evaluation of antiviral agents. This project will make the GBV-B antiviral assay widely available, affordable, and appropriate for HTP applications. This will facilitate the discovery and development of new anti-HCV drugs that may improve the quality of life for HCV-infected patients.